Looking through the imaging perspective: the importance of imaging necrosis in glioma diagnosis and prognostic prediction – single centre experience

Abstract Background The aim of the study was to investigate the diagnostic value of imaging necrosis (Imnecrosis) in grading, predict the genotype and prognosis of gliomas, and further assess tumor necrosis by dynamic contrast-enhanced MR perfusion imaging (DCE-MRI). Patients and methods We retrospectively included 150 patients (104 males, mean age: 46 years old) pathologically proved as adult diffuse gliomas and all diagnosis was based on the 2021 WHO central nervous system (CNS) classification. The pathological necrosis (Panecrosis) and gene mutation information were collected. All patients underwent conventional and DCE-MRI examinations and had been followed until May 31, 2021. The Imnecrosis was determined by two experienced neuroradiologists. DCE-MRI derived metric maps have been post-processed, and the mean value of each metric in the tumor parenchyma, peritumoral and contralateral area were recorded. Results There was a strong degree of inter-observer agreement in defining Imnecrosis (Kappa = 0.668, p < 0.001) and a strong degree of agreement between Imnecrosis and Panecrosis (Kappa = 0.767, p < 0.001). Compared to low-grade gliomas, high-grade gliomas had more Imnecrosis (85.37%, p < 0.001), and Imnecrosis significantly increased with the grade of gliomas increasing. And Imnecrosis was significantly more identified in IDH-wildtype, 1p19q-non-codeletion, and CDKN2A/B-homozygous-deletion gliomas. Using multivariate Cox regression analysis, Imnecrosis was an independent and unfavorable prognosis factor (Hazard Ratio = 2.113, p = 0.046) in gliomas. Additionally, extravascular extracellular volume fraction (ve) in tumor parenchyma derived from DCE-MRI demonstrated the highest diagnostic efficiency in identifying Panecrosis and Imnecrosis with high specificity (83.3% and 91.9%, respectively). Conclusions Imnecrosis can provide supplementary evidence beyond Panecrosis in grading, predicting the genotype and prognosis of gliomas, and ve in tumor parenchyma can help to predict tumor necrosis with high specificity.


Introduction
2][3] Though the importance of necro-sis in gliomas has already been addressed, necrosis was first incorporated into the determinant of the diagnosis for glioma grade in the fifth edition of the 2021 WHO classification of Tumors of the central nervous system (CNS), which highlighted and underlined the significant value of necrosis in the diagnosis and prognosis of adult diffuse gliomas. 4According to the latest classification, once histological necrosis is identified, a diagnosis of WHO grade 4 astrocytoma or glioblastoma is suggested.However, there is a diagnostic dilemma in grading gliomas by identifying necrosis.
Presently, necrosis is primarily determined by pathological examination, in which partial tumor specimens from specific sites of tumors obtained by surgery or biopsy at a single point in time are generally inspected. 5However, due to tumor heterogeneity and incompleteness of the pathological sample, some pathological necrosis is likely to be missed, which may result in an underestimation of tumor grades, especially when the molecular analysis is not available.As tumor grades influence therapeutic decisions and prognosis, it is imperative to make up for the problem of a missed diagnosis of necrosis on pathological evaluation.
Magnetic resonance imaging (MRI) is utilized for routine, noninvasive, preoperative examination in diagnosing gliomas.Pathological necrosis usually has corresponding imaging features. 6,7maging necrosis has been defined as a region within the tumor that does not enhance or shows markedly diminished enhancement, high signal intensity on T2WI, low signal intensity on T1WI, and an irregular border. 6[10][11][12][13] Moreover, conventional and advanced MRI can acquire comprehensive morphological and pathophysiological images of entire tumors, which is impossible with pathological examinations.
Taking all of this into account, we speculated whether necrosis diagnosed by MRI (hereafter termed "imaging necrosis", abbreviated as Im necrosis ) could be used as a correction or a supplement to necrosis diagnosed by pathological evaluation (hereafter termed "pathological necrosis", abbreviated as Pa necrosis ), especially when there is no evidence of Pa necrosis owing to limited sampling sites and sampling amounts.Consequently, herein, we retrospectively reviewed MRI findings of adult diffuse gliomas that were diagnosed based on the 2021 WHO CNS classification and assessed the role of Im necrosis in grading, predicting the genotype and prognosis of gliomas.We also attempted to analyse tumor necrosis by dynamic contrastenhanced MR perfusion imaging (DCE-MRI) to validate quantitative imaging markers for probing tumor necrosis.

Study participants
Patients with a primary diagnosis of glioma (June 2013-May 2021) were retrospectively included.Inclusion and exclusion criteria are presented in Supplementary Figure 1.Clinical information of patients was retrieved from the electronic medical records, and follow-up information was obtained through clinical interviews.Follow-up survival data were available until May 31, 2021.Overall survival (OS) was calculated from the initial surgery date to the date of death, or the date of the last follow-up visit if the patient was alive or lost to follow-up.
This retrospective analysis was in accordance with the ethical standards of the institutional and national research committee and was approved by the ethics committee of our institution ([2021]209).The requirement for written informed consent was waived due to the retrospective nature of this study.

MRI parameters
Participants underwent conventional (T1/T2weighted images [T1WI/T2WI], T2-weighted fluidattenuated inversion recovery [T2WI-FLAIR] and sagittal view of contrast-enhanced three-dimensional T1 MPRAGE images) and DCE-MRI imaging using a 3.0T MR system (Magnetom Verio, Siemens Medical Solutions, Erlangen, Germany) with a 64-channel head-neck coil.The parameter details of the conventional MRI and the DCE-MRI were elaborated in Supplementary Appendix 1.

Image processing
All DCE-MRI data were transferred to the postprocessing workstation (detailed in Supplementary Appendix 2).Pharmacokinetic parameters, including the transfer constant (ktrans), extravascular extracellular volume fraction (ve), rate constant (kep = ktrans/ve), and initial area under the curve in the first 60 s (iauc), were automatically generated.Regions of interest (ROIs) were selected across three consecutive maximum tumor parenchyma slices.At each slice, one ROI was put in tumor parenchyma (hereafter termed "tumor"), according to T2WI-FLAIR, and enhanced T1WI, avoiding necrosis, cystic, and vessel areas.Another two approximate 2-mm-diameter ROIs were put in tumor peripheral zones (hereafter termed "edema", within a 1-cm distance from the outer enhancing tumor margin) and contralateral normal-appearing brain tissues (hereafter termed "control") (Figure 1).The mean values of each DCE-MRI metric was recorded.
As mentioned in the introduction, examples of imaging necrosis, defined as a region within the tumor that does not enhance or shows markedly diminished enhancement, high signal intensity on T2WI, low signal intensity on T1WI, and an irregular border, are shown in Figure 2 and Supplementary Figure 2. Two experienced radiologists reviewed all conventional MRIs.Then they determined whether there was Im necrosis by consensus.One of these two experienced radiologists and a third radiologist repeatedly assessed 68 cases after the initial assessment to assess the inter-observer agreement.The assessed images were randomized within each type of pathology, and the observers were blinded to the clinical and pathological information and thoroughly acquainted with the criteria.

Pathological and molecular analysis
Pa necrosis was defined according to pathological reports provided by the Pathology department of our hospital, if available.The status of 1p19q codeletion, EGFR amplification, chr7 gain/10 loss (+7/-10), and CDKN2A/B homozygous deletion were determined by fluorescence in situ hybridization with a specific probe.IDH mutation was determined by high-throughput sequencing, including IDH1 and IDH2 mutations.The pathological diagnosis and grading of gliomas were reassigned according to the 2021 WHO CNS classification (Supplementary Figure 3). 4,14,15

Statistical analysis
Statistical Analysis Data were analyzed using IBM SPSS Statistics 26 software, the SPSSAU data scientific analysis platform (https://spssau.com/),and the R programming language (version 4.1.2,The R Foundation for Statistical Computing).Normally distributed continuous variables were compared using unpaired t-tests, whereas non-parametric tests were used for non-normally distributed variables.Descriptive data are expressed as mean ± SD, except where otherwise stated.Unpaired t-tests, non-parametric tests, and chi-squared tests were used to compare differences between parameters.Receiver Operating Characteristics (ROC) curves

Patients' demographic and clinical findings
We initially identified 150 eligible patients (median age = 46 years, range 21-79 years), and 104 (69.33%) were male (Table 1).All the diagnoses assigned to the patients according to the latest integrated histo-molecular classification criterion were presented in Supplementary Figure 3 and

Interobserver agreement of imaging necrosis and correlation between imaging and pathological necrosis
In this study, the following four groups were determined: Im+Pa  2. We found strong agreement between Im necrosis and Pa necrosis (Kappa = 0.767, p < 0.001, 95%CI: 0.637-0.897).
Significant differences in the presence of Im necrosis with a large effect size were found between HGGs and LGGs and among different grades of gliomas (Table 1, p < 0.001).Cochran-Armitage tests showed an upward trend in Im necrosis from lower to higher grades of gliomas (p < 0.001).Multiple comparisons with Bonferroni correction showed that the difference between WHO grades (any two grades) and Im necrosis was significant (all p < 0.01).32.08%, 85.94% vs. 51.11%,20% vs. 0, respectively).However, no significant correlation was found between Im necrosis and EGFR amplification or +7/-10 cytogenetic signature (p > 0.05) (Table 1).

Association of imaging necrosis with patient prognosis
One-hundred and thirty patients were included in the final survival analysis.Compared with gliomas with Im necrosis , patients without Im necrosis had a significantly longer survival time (p < 0.001, Figure 4A).By reference to the gliomas with Pa necrosis , patients without Pa necrosis had a sig-nificantly longer survival time as well (p < 0.001, Figure 4B).The differences among the OS of Im+Pa necrosis , no necrosis , Only Im necrosis , and Only Pa necrosis groups were statistically significant (p < 0.01, Figure 4C).Further, after Bonferroni correction, there were significant differences between Im+Pa necrosis and no necrosis groups (p < 0.001).According to Figure 4C, the OS of the Only Pa necrosis group (n = 2) was shorter than the OS of no necrosis group (n = 28) and Only Im necrosis group (n = 7).Between the two survival curves of no necrosis and the Only Im necrosis groups, there were marked crossovers, but within a certain period (time spanning about from 5-month

Correlation of tumor necrosis and DCE-MRI metrics
Since pathology is the golden standard for necrosis diagnosis, we analyzed the associations with Pa necrosis and DCE-MRI metrics.Most DCE-MRI metrics demonstrated a significant difference in identifying gliomas with Pa necrosis with a very large effect size (Table 3).Kep was significantly higher for gliomas with Pa necrosis than those without Pa necrosis , while other DCE-MRI metrics showed the opposite trend.ROCs analysis showed that the Tumor-ve-Mean displayed the best diagnostic performance with the largest AUC of 0.891 (95%CI: 0.788-0.995,p < 0.0001), and the optimal cut-off point was 0.17 with a sensitivity of 96% and specificity of 83.3%.
Similarly, we performed the analysis regarding Im necrosis (Table 3), and the Tumor-ve-Mean displayed the best diagnostic performance as well, with the most significant AUC of 0.929 (95%CI: 0.872-0.986,p < 0.0001) and the optimal cut-off point was 0.17 with a sensitivity of 89.2% and specificity of 91.9%.

Discussion
In this study, we investigated the clinical implication of imaging necrosis in the preoperative evaluation of glioma.We found strong agreement between Im necrosis and Pa necrosis .Moreover, Im necrosis was found to be significantly related to gliomarelated key gene mutations, such as 1p19q noncodeletion and CDKN2A/B homozygous deletion.And it is an independent imaging marker for predicting tumor prognosis.Additionally, tumor parenchyma ve derived from DCE-MRI can help to predict tumor necrosis with high specificity.
Our study indicated strong agreement between the inter-observer agreement of Im necrosis and Pa necrosis .And during the analysis, we found that (A) a 24-year-old man with an oligodendroglioma, IDH-mutant and 1p/19q-deleted, CNS WHO grade 2; (B) a 39-year-old man with an oligodendroglioma, IDH-mutant and 1p/19q-deleted, CNS WHO grade 2; (C) a 55-year-old woman with an oligodendroglioma, IDHmutant and 1p/19q-deleted, CNS WHO grade 2; (D) a 45-year-old man with an astrocytoma, CNS IDH-mutant, WHO grade 2; (E) a 36-year-old woman with an oligodendroglioma, IDH-mutant and 1p/19q-deleted, CNS WHO grade 2. In this case (E), it showed multiple long tubular and filiform enhancement and there were some tumor areas with remarked decrease of reinforcement.But these areas are hyperintense, not hypointense, on the T1-weighted image.Comparing with CT images (not provided), calcification on these areas were just observed.So, there was no imaging necrosis in these conditions.
the regions with an absence or marked decrease of enhancement inside the intensified areas were easily mistaken as Im necrosis .While considering pathological samples were partial, imaging observation can capture full tumors.There was a pathologically proven astrocytoma, IDH-mutant, CNS WHO grade 2, with a very short OS (5 months).We reviewed the raw data and identified that this patient had a small extent of Im necrosis , indicating high grade gliomas.The situation mentioned above can be avoided if a judgement of Im necrosis is made, which is one unique advantage of radiographic examination.Besides, we identified seven patients with Im necrosis who were diagnosed as oligodendrogliomas, IDH-mutant and 1p/19q-deleted, CNS WHO grade 2 or 3, indicating that necrosis plays a limited predictive value in oligodendrogliomas.Hence, if there is evidence of oligodendrogliomas, such as calcification and filiform or localized internal homogeneous enhancement, Im necrosis does not indicate a high-grade tumor.Besides, Waqar et al. reported this kind of reinforcement as a "chicken wire" appearance with the explanation that oligodendroglioma vasculature often was described as a network of regular fine branching capillaries. 16,179][20] Our results were in accordance with their findings.Besides, the latest WHO CNS classification emphasizes the role of molecular markers, such as IDH, 1p19q, CDKN2A/B, 7+/10-, and EGFR, in the diagnosis and prediction of the prognosis of gliomas. 4From this prospect, Im necrosis might be more critical than Pa necrosis since it can be non-invasively obtained before operation.However, there was no significant difference between the expressions of 7+/10-cytogenetic signature or EGFR amplification and the presence of Im necrosis .This negative result might be due to the small sample size and insufficient number of events.
In this study, we also sought quantitative metrics for indicating tumor necrosis.][23] Hence, DCE-MRI metrics, especially ve in tumor parenchyma (cut-off value: 0.17), might be a supplementary metric to the morphological observation for delineating tumor necrosis.
The current study has some limitations.First, since evidence of pathological necrosis was obtained from pathology reports of the same hospital, there may be an observation bias.However, this study, based on clinical real-world evidence, can exactly address the current clinical deficits.Second, this is a single-center study; subgroups analysis had a small sample, which might result in insufficient power to reach definite conclusions.Further multicenter studies with large sample sizes will help improve the efficacy of Im necrosis in predicting the expression of molecular markers and prognosis.

Conclusions
Based on the latest WHO CNS guidelines, the present study depicted the importance of imaging necrosis in diagnosing gliomas.Detection of imaging necrosis in gliomas probably suggests an HGG unless there is imaging evidence for oligodendrogliomas, IDH-mutant and 1p/19q-deleted.Imaging necrosis was significantly associated with glioma-related key gene mutations, such as 1p19q non-codeletion and CDKN2A/B homozygous deletion.And it is an independent imaging marker for predicting tumor prognosis.Additionally, the Tumor-ve-Mean derived from DCE-MRI can help to predict necrosis with high sensitivity and specificity.Overall, in this study, we re-evaluated the imaging necrosis in the assessment of gliomas and provided a feasible solution to solve the frequent diagnostic dilemma of gliomas.
used to evaluate diagnostic efficacy.Simple kappa was calculated to assess the consistency of different diagnoses and inter-observer agreement.Kaplan-Meier survival analysis was used to analyze survival data.Hazard ratios (HR) were estimated according to the Cox proportional hazard method.A two-sided p value < 0.05 was considered significant.Detailed statistical methods are shown in Supplementary Appendix 3.
necrosis group (representing patients with both Im necrosis and Pa necrosis , n = 74), no necrosis group (representing patients without Im necrosis nor Pa necrosis , n = 28), Only Im necrosis group (representing patients with Im necrosis but without Pa necrosis , n = 7), and Only Pa necrosis group (representing patients with Pa necrosis but without Im necrosis , n = 4) groups.Detailed clinical, imaging and paychological information of Only Im necrosis group and Only Pa necrosis group were shown in Table

FIGURE 2 .
FIGURE 2. Representative MR images with imaging necrosis derived from a 53-year-old man with glioblastoma, IDH-wildtype.Shown from left to right by the order are T1WI (A), T2WI (B), and T1WI-CE (C).

FIGURE 3 .
FIGURE 3. Some representative MRI images without imaging necrosis which was exactly confused in diagnosing imaging necrosis.Shown from left to right by the order are T1WI, T2WI, and T1WI-CE.(A) a 24-year-old man with an oligodendroglioma, IDH-mutant and 1p/19q-deleted, CNS WHO grade 2; (B) a 39-year-old man with an oligodendroglioma, IDH-mutant and 1p/19q-deleted, CNS WHO grade 2; (C) a 55-year-old woman with an oligodendroglioma, IDHmutant and 1p/19q-deleted, CNS WHO grade 2; (D) a 45-year-old man with an astrocytoma, CNS IDH-mutant, WHO grade 2; (E) a 36-year-old woman with an oligodendroglioma, IDH-mutant and 1p/19q-deleted, CNS WHO grade 2. In this case (E), it showed multiple long tubular and filiform enhancement and there were some tumor areas with remarked decrease of reinforcement.But these areas are hyperintense, not hypointense, on the T1-weighted image.Comparing with CT images (not provided), calcification on these areas were just observed.So, there was no imaging necrosis in these conditions.

FIGURE 4 .
FIGURE 4. Survival curves for cases of imaging necrosis (A), cases of pathological necrosis (B), and cases of both pathological and imaging necrosis (C).
There were significant correlations between the expression of other molecular markers such as IDH, 1p19q, and CDKN2A/B and the presence of Im necrosis .According to Table1, the proportion of IDH-wildtype, 1p19q-non-codeletion, or CDKN2A/ B-positive cases with Im necrosis was significantly higher than that of cases without Im necrosis with a medium effect size, respectively (75.82% vs.

TABLE 1 .
Participant demographic findings & = Student's t statistic in this cell, and other cells in the same column represent Chi-square values.a and b = chi-square tests with continuity correction and Fisher'

TABLE 2 .
Detailed clinical, imaging and pathological information of Only Im necrosis group and Only Pa necrosis group CNS = central nervous system; NA = not available; NOS = not otherwise specified parenchyma, except kep, were significantly higher in gliomas with Im necrosis /Pa necrosis .And ve in tumor parenchyma demonstrated the highest diagnostic efficiency in identifying tumor necrosis with high sensitivity and specificity.Significantly high DCE-MRI metrics may be attributed to gliomas growing uncontrollably fast, resulting in severe hypoxia and necrosis.Thus, an extensively hyperpermeable vasculature is generated, resulting in inadequate oxygen and supplements delivery.The

TABLE 3 .
Representative results of non-parametric tests and ROC analyses between DCE-related data for gliomas with or without pathological necrosis/imaging necrosis * = p < 0.05; ** = p < 0.01